The long-term goal of this project is to elucidate the role(s) of protein phosphorylation in the regulation of the gamma-aminobutyric acid (GABA) receptor/channel complex, or GABAA receptor. This receptor/chloride ion channel is the primary target of GABA, the major inhibitory neuro- transmitter of the mammalian brain, and is the site of action of benzodiazepines and barbiturates used in the treatment of anxiety disorders and epilepsy. Phosphorylation of the receptor by intracellular protein kinases may alter the expression, modification and assembly of the receptor/channel complex, its receptor binding and channel gating characteristics, and/or its desensitization and degradation. A better understanding of the regulation of GABAA channel function by protein kinases may guide the development of new anticonvulsant and anxiolytic agents and influence the clinical treatment of seizure disorders. The proposed studies will employ and develop techniques in neurophysiology, neuropharmacology, and molecular biology to characterize whole cell and single channel currents generated by cloned GABAA receptors expressed in cultured cells. Plasmid constructs containing cDNAs encoding GABAA receptor subunits, and lacZ (the E. coli beta-galactosidase gene, used as a transfection marker), will be acutely co-transfected into cultured fibroblast (L929) cells. After identifying transfected cells with a fluorescent beta- galactosidase substrate, GABAA currents will be recorded using "whole cell" and single channel patch clamp recordings to study GABAA receptor pharmacology. Protein kinase A (catalytic subunit) and protein kinase C (catalytically active) will be included in the recording pipette solution and dialyzed or perfused through the electrode into the cell, or onto the cytoplasmic surface of an "outside-out" patch. GABA, benzodiazepines, barbiturates and neurosteroids will be applied by pressure ejection from a nearby pipette, or microperfused in the area of the cell or patch for rapid application and washout studies. Data will be digitized on-line and subsequently analyzed by computer programs to determine channel conductance states and gating kinetics, the kinetics of agonist-dependent desensitization and agonist-independent "rundown." Site-directed mutagenesis will be used to alter the primary structure of GABAA receptor subunits by mutating phosphorylation sites to determine precise structure/function relationships. Other cell types will be used to express GABAA receptors in larger quantities for receptor binding experiments to examine changes in receptor binding kinetics in response to desensitization or receptor phosphorylation states.